Defense Date


Document Type


Degree Name

Doctor of Philosophy



First Advisor

Clive Baumgarten


Swelling-activated Cl− current (ICl,swell) is an outwardly-rectifying current that plays an important role in cardiac electrical activity, cellular volume regulation, apoptosis, and acts as a potential effector of mechanoelectrical feedback. Persistent activation of ICl,swell has been observed in a number of models of cardiovascular disease. Previously we showed that angiotensin II (Ang II), endothelin-1 (ET-1), endothelial growth factor receptor (EGFR), and reactive oxygen species (ROS) produced by NADPH oxidase (NOX) and mitochondria are involved in the activation of ICl,swell by both osmotic swelling and Beta1 integrin stretch. Sphingolipid metabolism is modulated in several cardiopathologies and because sphingolipids are bioactive lipids involved in signaling cascades that overlap significantly with these modulators of ICl,swell, we investigated the role of sphingolipids in the regulation of ICl,swell in cardiac ventricular myocytes. Under isoosmotic conditions that isolate anions currents, addition of exogenous, cell permeant C2-ceramide (C2-Cer) elicited an outwardly-rectifying Cl− current that reversed near the Cl− equilibrium potential (ECl) in both physiological and symmetrical Cl− gradients. This current was inhibited by the ICl,swell-specific blockers DCPIB. Dihydro-C2-ceramide (C2-H2Cer), the inactive analogue of C2-Cer, failed to elicit current. These data strongly suggest that the identity of C2-Cer-induced Cl− current is ICl,swell and indicate that sphingolipid signaling pathways may be involved. Bacterial sphingomyelinase (SMase), which converts endogenous sphingomyelin in the outer leaflet of the sarcolemmal membrane to native chain-length ceramides, elicited a DCPIB-sensitive Cl− current. SMase-induced current is also suppressed by tamoxifen, which under conditions that isolate anion currents is a specific inhibitor of ICl,swell. SMase-induced ICl,swell was abrogated by ebselen, a membrane permeant glutathione peroxidase mimetic that dismutates H2O2 to H2O. This suggests that ROS are required mediators of SMase-induced activation of ICl,swell. Both NOX and mitochondria are important sources of ROS in cardiomyocytes and both have been implicated in modulating ICl,swell. Blocking NOX with apocynin or the NOX fusion peptide inhibitor gp91ds-tat had no effect on SMase-induced current. However, pretreatment of cardiomyocytes with gp91ds-tat reduced the maximum current amplitude of SMaseinduced ICl,swell, indicating that NOX may play a time-dependent role in this mechanism. By contrast, the mitochondrial Complex I blocker rotenone, which suppresses extramitochondrial ROS release by Complex III, completely suppresses SMase-induced ICl,swell. Additionally, SMase-induced ICl,swell is partially inhibited by blockade of mitochondrial KATP (mitoKATP) channels with 5-hydroxy-decanoic acid (5-HD). MitoKATP channels have been implicated as modulators of mitochondrial ROS release. Thus these data suggest that mitochondrial ROS generation is required for SMaseinduced activation of ICl,swell. Ceramides are metabolized to form several sphingolipids, including sphingosine-1-phosphate (S1P). We tested whether ceramide metabolites are responsible for eliciting ICl,swell. Under isosmotic conditions that isolate anion currents, SMase-induced ICl,swell was abrogated by blockade of ceramidase, which converts ceramide to sphingosine, with Derythro-MAPP. SMase-induced ICl,swell also was suppressed by inhibition of sphingosine kinase with DL-threo-dihydrosphingosine. These data suggested that the ceramide metabolite S1P is likely to stimulate ICl,swell. As expected, exogenous S1P elicited an outwardly rectifying Cl− current that was fully inhibited by DCPIB. As seen with SMaseinduced ICl,swell, S1P-induced ICl,swell was fully inhibited by rotenone. In contrast to results with SMase, S1P-induced current was partially inhibited by blockade of NOX with apocynin. These data indicate that S1P is a necessary component of SMase-induced ICl,swell activation and that the action of exogenous S1P involves ROS from both mitochondria and NOX. Importantly, the fact that exogenous C2-ceramide also activates ICl,swell even though C2-ceramide may not metabolized to S1P in native cells. Thus, it seems likely that ceramides can elicit ICl,swell via S1P and also by a distinct pathway and that both pathways converge at mitochondrial ROS. In order to determine the role of ERK in the proposed signaling pathway that regulates ICl,swell, we examined the effect of ERK inhibitors PD98059 and U0126 on the activation of ICl,swell. Both of these agents partially inhibited SMase-induced activation of ICl,swell, indicating SMase acts through both ERK-dependent and ERK-independent signaling pathways. HL-1 cells are derived from a murine atrial cell line that retains phenotypic characteristics of adult cardiomyocytes. Recently, ICl,swell has been observed in HL-1 cells with similar regulatory mechanisms to those seen in native cells. We showed that SMase elicits an outwardly-rectifying, DCPIB-sensitive Cl− current that reverses near ECl in HL-1 cells. Finally, we confirmed the production of ROS by SMase-induced signaling by flow cytometry in HL-1 cells using the nominally H2O2-selective fluorescent probe CH2DCFDA-AM. Exposure to SMase increased ROS production, as did the positive control H2O2. SMase-induced ROS generation was suppressed by pretreatment with rotenone but was unaffected by pretreatment with gp91ds-tat. These data indicate that exogenous and endogenous sphingolipids elicit ICl,swell in cardiomyocytes by stimulating mitochondrial ROS production. NOX may contribute to the ROS generation, but is not a required step in this mechanism. Sphingolipid signaling is likely to play an important role in stimulating ROS production and activating ICl,swell in a number of cardiovascular diseases.


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Date of Submission

December 2009

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